THROMBOSIS RESEARCH 66; 179-189,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon PressLtd.Allrights reserved.
RAPID QUANTITATIVE EVALUATION OF PLASMA D-DIMER LEVELS IN THROMBOTIC
STATES USING AN AUTOMATED
Kazuomj Kariolv 2, Matsuo .Katsuichirou
LATEX PHOTOMETRIC
IMMUNOASSAY
Takefumi M?tsuol, Hiroko KaQayashi3. Miyaka Yamamoto , George Sakurai , Mituhiro Baba
Department of Internal Medicine1 and Central laboratory3, Prefectural Awaji Hospital, Sumoto, Hyogo, 656-16. Japan, Chiba, 289-22, Iatron Laboratories, Research Division*,
Hyogo and Japan
(Received 21.6.1991; accepted in revised form 5.2.1992 by Editor J. Soria)
ABSTRACT To evaluate a recently developed latex photometric immunoassay (LPIA) that which can measure 40 samples quantitatively within 30 minutes, we measured D-dimer levels in blood samples obtained from patients with disseminated intravascular coagulation (DIG). Linearity of D-dimer determination was shown over the range from 0.5 to 36 fig/ml, and recovery studies demonstrated 94 to 108 % recovery. The intra-assay and inter-assay coefficients of variation ranged from 0.6 to 11.3 % at plasma D-dimer levels of 0.54 to 30.1 fig /ml. No interference by lipids, bi 1 irubin, haemogl obin, rheumatoid factor, or r-globulin was noted. The normal D-dimer range was < 0.5 fig/ml in healthy ambulatory subjects, while the level in elderly subjects with atherosclerosis (14 %) or in immobilized subjects (38 %) was well above this limit. There was a strong correlation between plasma and serum D-dimer levels (r=O. 993). D-dimer levels measured by this LPIA showed a good correlation with those determined using two kinds of ELISA. The LPIA D-dimer levels were elevated in some subjects with diseases predisposing to DIC, but remained below 10.0 fig/ml. On the other hand, the LPIA D-dimer levels in DIC subjects were almost always above 10.0 fig/ml. Our study showed that a hypercoagulable state should be suspected when the LPIA D-dimer level is > 0.5 /rg/ml and that DIC should be diagnosed when the level is > 10.0 Lrg/ml in the presence of an appropriate underlying disease. This LPIA system can rapidly evaluate the presence of a hypercoagulable state as and thus seems accurately as any ELISA, potentially valuable for both emergency and routine laboratory use. Key words: Latex photometric immunoassay, D-dimer, Elderly. Atherosclerosis, Disseminated intravascular CoaiWlation 179
180
D-DIMER IN THROMBOTIC STATES
Vol. 86, Nos. 2l3
INTRODUCTION D-dimer is a fragment that is specifically produced by fibrin rather than fibrinogen degradation, so its increase in fibrin formation and subsequent the blood indicates recent Enzyme-linked immunosorbent assay (ELISA) degradation (1). levels provides the best method for measuring D-dimer but the results are not available for at least quantitatively, which represents a serious handicap for screening in 4 hours, although the latex agthe emergency ward. On the other hand, it is less sensitive and glutination test is rapid and simple, tends to underestimate the D-dimer level. Thus, there is a poor correlation between the results of ELISA and the latex agglutination test, especially at low D-dimer levels (l-6). The recently developed latex photometric immunoassay (LPIA) method for various rapid and quantitative assay Using this system, we measured D-dimer levels ittigzns. (7,8) in 56 blood ‘samples obtained from patients with disseminated intravascular coagulation (DIG), and compared then with the levels in various control groups to assess haemostatic activity in the thrombotic state.
MATERIALS AND METHODS Technical report: D-dimer levels were determined by the following latex photometric immunoassay system (LPIA-100, Mitsubishi Kasei Co., Japan) which measured the increase in turbidity due to latex agglutination reaction. The reagents used for this LPIA were as follows: 1) The standard for calibration was the DD/E complex (0, 4, 8, 16, 32 fig/ml) isolated by the method of Olexa et al. (9) and dissolved in human serum containing aprotinin (1 trypsin inhibitor unit/ml). The DD/E complex level in the standard samples was spectroscopically quantified by the absorbance of a l-cm cell at 280nm, which was 18.4 for a 1 % DD/E complex solution. 2) The latex suspension was a borate buffer containing polyethylene latex particles (0.4 %) coated with mouse monoclonal anti D-Dimer antibody (JIF-23, Iatron. Japan) (10). The coating method was a modified version of that 7; lS;do et al. (7). 3) The buffer solution was Tris-HCl buffer pH 8. 2, ionic strength: 0.033). 4) The stabilizing solution was Tris-HCl buffer (0.2 M, pH 8. 2, ionic strength: 0.025) containing 0.5 96 bovine serum albumin. 5) The diluting solution was human serum containing aprotinin at 1 Trypsin inhibitor unit/ml. The following assay procedures were performed automatically. Test plasma (5 ~1) was dispensed with 60 ~1 of buffer solution and 200 ~1 of the stabilizing solution into the reaction cuvettes. After a lo-min stabilizing period at 37 “c, 40 ~1 of latex suspension was dispensed into the reaction cuvettes. Using a photometer designed for the LPIA which operated with near-infrared light of 950 nm. the increase in
Vol. 66, Nos. 213
D-DIMER IN THROMBOTIC STATES
181
turbidity was assessed every 15 set; a total of 40 measurements were performed during a lo-min reaction period at 37 “c. Using the calibrated reaction velocities of each of the 5 standards shown in Fig. 1. the D-dimer level of the test sample was calculated from its reaction velocity. Using this system, 40 samples could be assessed within 30 min, automatically. Clinical report: The serum and plasma samples used in this study were obtained from the patients with untreated DIC. Patients were studied who had appropriate underlying diseases and whose laboratory data fulfilled the criteria proposed by the Research Committee on DIC of the Ministry of Health and Welfare of Japan (11, 12). Briefly, they had prolongation of the prothrombin time, a decrease in the platelet count and plasma fibrinogen level, and elevation of the serum fibrin/fibrinogen degradation products (FDP) level. The diagnosis of DIC was confirmed by a positive fibrin monomer test and the elevation of thrombin-ATllI complex (TAT: > 4 fig/l) and plasmin-cu 2 antiplasmin complex (PAP; > 0.8 mg/l) levels. The underlying diseases included (n=15), cancer sepsis (n=13), acute promyelocytic leukemia (n=ll), acute myelocytic leukemia (ng5). vascular damage (n=3), head injury (n=2), trauma (n=4), and shock excluded.(n=3~iftyB~?$ ~~~~~~~ntfro~it~re~~~~~ly~~~ien~~se~~~~ predisposing to DIC (sepsis: n=15, cancer: n=22, trauma: n=5, shock : n=7, acute myelocytic leukemia: n=6) who did not have DIC, 50 healthy young volunteers under 50 years of age, 66 ambulant healthy elderly volunteers (mean age: 76.5k8.2 years), 63 elderly outpatients with atherosclerotic disease (mean age: 76. 9f7.7 years), and 21 immobilized elderly patients (mean age: 82.8+ 7.0 years) were also examined. Blood samples for assessing haemostatic parameters were collected into disposable siliconized glass tubes with 3.8 % trisodium citrate (9 vol. 0.13 M trisodium blood to 1 vol. citrate solution), 15 min at and centrifuged at 3,000 g for room temperature within 60 min. Plasma for the determination of TAT and PAP levels was subsequently separated and stored in Whole blood samples colplastic tubes at -80°C until analysis. lected in EDTA were processed to determine the platelet count within 1 hr. of collection using an automated analyzer (Technicon H*l). Serum for the FDP assay was collected into a special tube for FDP (No.705773901, The prothrombin Du Pont). time was measured by Quick’s one-stage method. Fibrinogen and FDP levels were determined by the fibrin polymerization method turbidimetric inhibition (13) and by a the particle-enhanced using an automated analyzer (Du immunoassay (14). respectively, Antithrombin IU (ATllI) and plasminogen levels were Pont aca). determined using amidolytic automated substrates and D-dimer was also detz:mined analyzer (Du Pont) (15). by two ELISA methods (1-Dimertest EIA. Mabco Ltd., Australia, 2France) (16-18). Plasma Asserachrom D-Di. Diagnostica Stage, TAT levels (Enzygnost-TAT, Behringwerke, Marburg, FRG, ) and PAP \;;eis,) (Tei jin Co., Tokyo, Japan) were measured by ELISA Soluble fibrin monomer complexes were detected by the hemagglutination technique (FS test. Diagnostica Stage) (21).
182
Vol. 66, Nos. 213
D-DIMER IN THROMBOTIC STATES
Statistical analysis: The data for D-dimer, TAT, PAP, and FDP were log-transformed. One-way analysis of variance and Welch’s t-test were used for comparison of the mean values in the various groups. In addition, correlation coefficients were calculated for the different variables.
RESULTS Evaluation of the latex photometric immunoassay system (LPIA) for D-dimer: Linearity of D-dimer levels was shown over the Recovery studies using from 0.5 to 36 fig/ml (Fig. 2). range three plasma samples with 8.2 or 16.1 fig/ml of D-dimer standard added showed from 94 to 108 % recovery (Table 1). No interrheumatoid factor, ference by lipids, bi 1 irubin, haemoglobin, The intra-assay and interor 7 -globulin was noted (Table 2). variation for the assessment of plasma samples from assay in Table patients with DIC using the LPIA system is presented Reproducibility was worse at low D-dimer levels compared 3. with at high D-dimer levels. There was a significant correlation between the plasma D-dimer (Y: mean 7.63 buff/ml) and serum D-dimer levels (X: 7.07 fig/ml) (Y = 1.101 X - 0.176. r = plasma levels exceeded serum 0.993. n = 50). In most subjects, levels. Correlations with the two ELISA tests are shown in Fig. 3. D-dimer levels obtained with the LPIA showed a good correlation with those determined by ELISA.
40
4
8 LPIA
16 D-dimer
1
32 (/q/ml) Dilution
FIG. Standard
curve
FIG. 2
1 for
the
LPIA
Dilution
test
Vol. 66, Nos. 2l3
D-DIMER IN THROMBOTIC STATES
Recovery
TABLE 1 Study of Three Plasma Samples Using the LPIA
D-dimer
level
Amount of standard D-dimer added
(bg/ml)
A
0.1
B
:*i 5:8 10.4 10.4
c
183
Recovery
(fig/ml)
6) 98. 2 107.6 94. 1 99. a 94. 9 98. 2
a. 2 16. 1 a. 2 16. 1 a. 2 16. 1
TABLE 2 Interference Study of the LPIA D-dimer
level
Amount of standard D-dimer added
(Yg/ml) A B c D E
A
(%)
(fig/ml) 8. 1 16. 4 a. i 16. 4 a. 1 16. 4 8. 1 16. 4 a. 1 16. 4
0.12 0.12 0.27 0. 27
RAPID QUANTITATIVE EVALUATION OF PLASMA D-DIMER LEVELS IN THROMBOTIC
STATES USING AN AUTOMATED
Kazuomj Kariolv 2, Matsuo .Katsuichirou
LATEX PHOTOMETRIC
IMMUNOASSAY
Takefumi M?tsuol, Hiroko KaQayashi3. Miyaka Yamamoto , George Sakurai , Mituhiro Baba
Department of Internal Medicine1 and Central laboratory3, Prefectural Awaji Hospital, Sumoto, Hyogo, 656-16. Japan, Chiba, 289-22, Iatron Laboratories, Research Division*,
Hyogo and Japan
(Received 21.6.1991; accepted in revised form 5.2.1992 by Editor J. Soria)
ABSTRACT To evaluate a recently developed latex photometric immunoassay (LPIA) that which can measure 40 samples quantitatively within 30 minutes, we measured D-dimer levels in blood samples obtained from patients with disseminated intravascular coagulation (DIG). Linearity of D-dimer determination was shown over the range from 0.5 to 36 fig/ml, and recovery studies demonstrated 94 to 108 % recovery. The intra-assay and inter-assay coefficients of variation ranged from 0.6 to 11.3 % at plasma D-dimer levels of 0.54 to 30.1 fig /ml. No interference by lipids, bi 1 irubin, haemogl obin, rheumatoid factor, or r-globulin was noted. The normal D-dimer range was < 0.5 fig/ml in healthy ambulatory subjects, while the level in elderly subjects with atherosclerosis (14 %) or in immobilized subjects (38 %) was well above this limit. There was a strong correlation between plasma and serum D-dimer levels (r=O. 993). D-dimer levels measured by this LPIA showed a good correlation with those determined using two kinds of ELISA. The LPIA D-dimer levels were elevated in some subjects with diseases predisposing to DIC, but remained below 10.0 fig/ml. On the other hand, the LPIA D-dimer levels in DIC subjects were almost always above 10.0 fig/ml. Our study showed that a hypercoagulable state should be suspected when the LPIA D-dimer level is > 0.5 /rg/ml and that DIC should be diagnosed when the level is > 10.0 Lrg/ml in the presence of an appropriate underlying disease. This LPIA system can rapidly evaluate the presence of a hypercoagulable state as and thus seems accurately as any ELISA, potentially valuable for both emergency and routine laboratory use. Key words: Latex photometric immunoassay, D-dimer, Elderly. Atherosclerosis, Disseminated intravascular CoaiWlation 179
180
D-DIMER IN THROMBOTIC STATES
Vol. 86, Nos. 2l3
INTRODUCTION D-dimer is a fragment that is specifically produced by fibrin rather than fibrinogen degradation, so its increase in fibrin formation and subsequent the blood indicates recent Enzyme-linked immunosorbent assay (ELISA) degradation (1). levels provides the best method for measuring D-dimer but the results are not available for at least quantitatively, which represents a serious handicap for screening in 4 hours, although the latex agthe emergency ward. On the other hand, it is less sensitive and glutination test is rapid and simple, tends to underestimate the D-dimer level. Thus, there is a poor correlation between the results of ELISA and the latex agglutination test, especially at low D-dimer levels (l-6). The recently developed latex photometric immunoassay (LPIA) method for various rapid and quantitative assay Using this system, we measured D-dimer levels ittigzns. (7,8) in 56 blood ‘samples obtained from patients with disseminated intravascular coagulation (DIG), and compared then with the levels in various control groups to assess haemostatic activity in the thrombotic state.
MATERIALS AND METHODS Technical report: D-dimer levels were determined by the following latex photometric immunoassay system (LPIA-100, Mitsubishi Kasei Co., Japan) which measured the increase in turbidity due to latex agglutination reaction. The reagents used for this LPIA were as follows: 1) The standard for calibration was the DD/E complex (0, 4, 8, 16, 32 fig/ml) isolated by the method of Olexa et al. (9) and dissolved in human serum containing aprotinin (1 trypsin inhibitor unit/ml). The DD/E complex level in the standard samples was spectroscopically quantified by the absorbance of a l-cm cell at 280nm, which was 18.4 for a 1 % DD/E complex solution. 2) The latex suspension was a borate buffer containing polyethylene latex particles (0.4 %) coated with mouse monoclonal anti D-Dimer antibody (JIF-23, Iatron. Japan) (10). The coating method was a modified version of that 7; lS;do et al. (7). 3) The buffer solution was Tris-HCl buffer pH 8. 2, ionic strength: 0.033). 4) The stabilizing solution was Tris-HCl buffer (0.2 M, pH 8. 2, ionic strength: 0.025) containing 0.5 96 bovine serum albumin. 5) The diluting solution was human serum containing aprotinin at 1 Trypsin inhibitor unit/ml. The following assay procedures were performed automatically. Test plasma (5 ~1) was dispensed with 60 ~1 of buffer solution and 200 ~1 of the stabilizing solution into the reaction cuvettes. After a lo-min stabilizing period at 37 “c, 40 ~1 of latex suspension was dispensed into the reaction cuvettes. Using a photometer designed for the LPIA which operated with near-infrared light of 950 nm. the increase in
Vol. 66, Nos. 213
D-DIMER IN THROMBOTIC STATES
181
turbidity was assessed every 15 set; a total of 40 measurements were performed during a lo-min reaction period at 37 “c. Using the calibrated reaction velocities of each of the 5 standards shown in Fig. 1. the D-dimer level of the test sample was calculated from its reaction velocity. Using this system, 40 samples could be assessed within 30 min, automatically. Clinical report: The serum and plasma samples used in this study were obtained from the patients with untreated DIC. Patients were studied who had appropriate underlying diseases and whose laboratory data fulfilled the criteria proposed by the Research Committee on DIC of the Ministry of Health and Welfare of Japan (11, 12). Briefly, they had prolongation of the prothrombin time, a decrease in the platelet count and plasma fibrinogen level, and elevation of the serum fibrin/fibrinogen degradation products (FDP) level. The diagnosis of DIC was confirmed by a positive fibrin monomer test and the elevation of thrombin-ATllI complex (TAT: > 4 fig/l) and plasmin-cu 2 antiplasmin complex (PAP; > 0.8 mg/l) levels. The underlying diseases included (n=15), cancer sepsis (n=13), acute promyelocytic leukemia (n=ll), acute myelocytic leukemia (ng5). vascular damage (n=3), head injury (n=2), trauma (n=4), and shock excluded.(n=3~iftyB~?$ ~~~~~~~ntfro~it~re~~~~~ly~~~ien~~se~~~~ predisposing to DIC (sepsis: n=15, cancer: n=22, trauma: n=5, shock : n=7, acute myelocytic leukemia: n=6) who did not have DIC, 50 healthy young volunteers under 50 years of age, 66 ambulant healthy elderly volunteers (mean age: 76.5k8.2 years), 63 elderly outpatients with atherosclerotic disease (mean age: 76. 9f7.7 years), and 21 immobilized elderly patients (mean age: 82.8+ 7.0 years) were also examined. Blood samples for assessing haemostatic parameters were collected into disposable siliconized glass tubes with 3.8 % trisodium citrate (9 vol. 0.13 M trisodium blood to 1 vol. citrate solution), 15 min at and centrifuged at 3,000 g for room temperature within 60 min. Plasma for the determination of TAT and PAP levels was subsequently separated and stored in Whole blood samples colplastic tubes at -80°C until analysis. lected in EDTA were processed to determine the platelet count within 1 hr. of collection using an automated analyzer (Technicon H*l). Serum for the FDP assay was collected into a special tube for FDP (No.705773901, The prothrombin Du Pont). time was measured by Quick’s one-stage method. Fibrinogen and FDP levels were determined by the fibrin polymerization method turbidimetric inhibition (13) and by a the particle-enhanced using an automated analyzer (Du immunoassay (14). respectively, Antithrombin IU (ATllI) and plasminogen levels were Pont aca). determined using amidolytic automated substrates and D-dimer was also detz:mined analyzer (Du Pont) (15). by two ELISA methods (1-Dimertest EIA. Mabco Ltd., Australia, 2France) (16-18). Plasma Asserachrom D-Di. Diagnostica Stage, TAT levels (Enzygnost-TAT, Behringwerke, Marburg, FRG, ) and PAP \;;eis,) (Tei jin Co., Tokyo, Japan) were measured by ELISA Soluble fibrin monomer complexes were detected by the hemagglutination technique (FS test. Diagnostica Stage) (21).
182
Vol. 66, Nos. 213
D-DIMER IN THROMBOTIC STATES
Statistical analysis: The data for D-dimer, TAT, PAP, and FDP were log-transformed. One-way analysis of variance and Welch’s t-test were used for comparison of the mean values in the various groups. In addition, correlation coefficients were calculated for the different variables.
RESULTS Evaluation of the latex photometric immunoassay system (LPIA) for D-dimer: Linearity of D-dimer levels was shown over the Recovery studies using from 0.5 to 36 fig/ml (Fig. 2). range three plasma samples with 8.2 or 16.1 fig/ml of D-dimer standard added showed from 94 to 108 % recovery (Table 1). No interrheumatoid factor, ference by lipids, bi 1 irubin, haemoglobin, The intra-assay and interor 7 -globulin was noted (Table 2). variation for the assessment of plasma samples from assay in Table patients with DIC using the LPIA system is presented Reproducibility was worse at low D-dimer levels compared 3. with at high D-dimer levels. There was a significant correlation between the plasma D-dimer (Y: mean 7.63 buff/ml) and serum D-dimer levels (X: 7.07 fig/ml) (Y = 1.101 X - 0.176. r = plasma levels exceeded serum 0.993. n = 50). In most subjects, levels. Correlations with the two ELISA tests are shown in Fig. 3. D-dimer levels obtained with the LPIA showed a good correlation with those determined by ELISA.
40
4
8 LPIA
16 D-dimer
1
32 (/q/ml) Dilution
FIG. Standard
curve
FIG. 2
1 for
the
LPIA
Dilution
test
Vol. 66, Nos. 2l3
D-DIMER IN THROMBOTIC STATES
Recovery
TABLE 1 Study of Three Plasma Samples Using the LPIA
D-dimer
level
Amount of standard D-dimer added
(bg/ml)
A
0.1
B
:*i 5:8 10.4 10.4
c
183
Recovery
(fig/ml)
6) 98. 2 107.6 94. 1 99. a 94. 9 98. 2
a. 2 16. 1 a. 2 16. 1 a. 2 16. 1
TABLE 2 Interference Study of the LPIA D-dimer
level
Amount of standard D-dimer added
(Yg/ml) A B c D E
A
(%)
(fig/ml) 8. 1 16. 4 a. i 16. 4 a. 1 16. 4 8. 1 16. 4 a. 1 16. 4
0.12 0.12 0.27 0. 27
